The overall objective of this project is to discover the molecular and structural mechanisms by which the mitotic apparatus moves chromosomes during mitosis. Our major approach is to develop an isolated mitotic apparatus model system that retains life-like microtubule assembly-disassembly characteristics and the capacity to move chromosomes. We have developed a method for mass isolating and storing mitotic cytoskeletons (membrane-free components of the mitotic apparatus) from sea urchin eggs and PtK1 tissue culture cells using a simple calcium-chelating, low-ionic strength, lysis buffer: "5-EMP" (5 mM EGTA, 0.5 mM MgCl2, and 10 mM PIPES, pH 6.8) plus 1% Nonidet P-40 non-ionic detergent and 20% glycerol to stabilize the microtubules. We have obtained, for the first time, clear evidence that spindle microtubules are depolymerizable by physiological concentrations (1-8 micro moles) of free calcium ions. Perfusion of metaphase and anaphase mitotic cytoskeletons with buffers that contain 1-2 mM ATP as well as 1-8 micro moles free calcium ions induced an apparent shortening of the chromosomal spindle fibers and, often, concurrent poleward movement of the chromosomes at physiological rates (1-6 microns/min). Although the 5-EMP isolates are not sensitive to depolymerization by cooling, hydrostatic pressure, or lack of exogenous tubulin, these features of spindle assembly in vivo are largely recovered after incubation of the isolated mitotic cytoskeletons in 5-EMP with 50% glycerol plus 10 micromoles EDTA. Ultrastructural and biochemical analyses of the mitotic cytoskeletons have revealed the presence of actin filaments (S1 labelling) laterally associated with the microtubules and high molecular weight proteins that co-migrate with egg dyneins (SDS-PAGE) and exhibit ATPase activity characteristic of dynein.